Fig. 4. Rab5 is part of a macromolecular complex containing Vps34 and beclin 1. (a) Colocalisation of endogenous Rab5 (green) and endogenous beclin 1 (red) in 3MA-treated HeLa cells after saponin extraction. DAPI-stained nuclei are in blue. (b) Colocalisation of constitutive active Rab5 (CA-Rab5; green) with wild-type beclin 1 (WT-Beclin, red). (c) COS-7 cells transfected with control vector alone (lane 1) or Flag-tagged wild-type beclin 1 alone (lane 2) or wild-type beclin 1 with wild-type Vps34 (lane 3) were immunoprecipitated using anti-Flag antibody (to immunoprecipitate beclin 1) and blotted for Rab5 using an anti-Rab5 antibody. (d) Q74-HA aggregation in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 as described in Fig. 2d; P<0.005. (e) Ratio of Atg5-Atg12 conjugate to Atg12 (data from three independent experiments) in HeLa cells transfected with control siRNA or siRNA targeting beclin 1 for 72 hours. Atg5 and HA-Atg12 were transfected for the last 24 hours. P=0.0003.